AMERICAN JOURNAL OF EPIDEMIOLOGY
Vol. 103, No
Copyright © 1976 by The Johns Hopkins University School of Hypene and Public Health
1
Printed mil SA
SOME BACTERIOLOGIC ASPECTS OF THE EPIDEMIOLOGY OF PULMONARY AND EXTRAPULMONARY TUBERCULOSIS1 2 SALMAN H. SIDDIQI, JOHN C. STAUFFER, MIR ABID ALI AND GARDNER MIDDLEBROOK
Although both catalase-positive and catalase-negative isoniazid-resistant strains of M. tuberculosis were isolated from patients with pulmonary disease, no catalase-negative strains were isolated from patients with extrapulmonary disease, suggesting limited pathogenic potentialities of catalase-negative strains for man. Epidemiologic aspects of these observations are discussed. catalase; drug resistance, microbial; tuberculosis; virulence INTRODUCTION
Numerous studies have been reported on drug resistance to tubercle bacilli (MycoReceived for publication July 31, 1975. Abbreviation INH, isoniazid, PAS, para-aminosalicylic acid, PPD-S, purified protein derivative of
bacterium tuberculosis) from patients in United States (1-10), the United Kingd o m (n~1^' a n d i n P a r t s o f t h e w o r l d where the incidence of tuberculosis is still very high (14-18). These studies have been devoted almost exclusively to pulmonary
t h e
,
. .
• ,
•• ,
P
tuberculin-standard tuberculosis, with little concern for ex1 From the Pakistan Medical Research Center, trapulmonary forms of the disease, probaUniversity of Maryland International CenterforMed- b j b e c a u s e human-to-human transmisical Research & Training, 6, Birdwood Road, Lahore, s l 0 n . , , Pakistan, and the Department of Pathology, Univer»S by the aerogenic route from cases sity of Maryland School of Medicine, Baltimore, MD with respiratory tract lesions. No report 21 ^1. , , , , , T - „ ,,. has been cited in the literature which deals This study was supported by the US Public ., Health Service, PL-480 Grant 08-009-1, Mycobacte- w l t n a comparative study of pulmonary nal Diseases in Pakistan. and extrapulmonary tuberculosis in gen101
Downloaded from https://academic.oup.com/aje/article-abstract/103/1/101/151952 by Tulane University user on 18 January 2019
Siddiqi. S. H . J . C. Stauffer, M. Ali and G. Middlebrook (Dept. Pathology, U. of Maryland School of Medicine. Baltimore. M D 21201). Some bacteriologic aspects of the epidemiology of pulmonary and extrapulmonary tuberculosis. Am J Epidemiol 103: 101-111, 1976. A study was carried out to investigate the drug resistance patterns of the prevalent tubercle bacilli in pulmonary and extrapulmonary tuberculosis in and about the city of Lahore, Pakistan. This report includes 168 strains of Mycobacterium tuberculosis isolated from the same number of pulmonary tuberculosis cases (100 untreated cases, defined as patients either having no history of anti-tuberculous therapy or having had chemotherapy for not more than 10 days; 68 treated, defined as having had chemotherapy for more than 10 days), and 162 strains from the same number of extrapulmonary tuberculosis cases (77 untreated, 38 treated and 47 doubtful). The proportion method of drug susceptibility assay was employed. According to the procedures used in this study and with 1 % as the critical proportion for resistance, bacterial resistance was found to be very prevalent in pulmonary tuberculosis. Even among those cases in which no history of previous treatment was elicited, 46% were found to be excreting populations of tubercle bacilli having some degree of resistance to one or more of the primary drugs—isoniazid, streptomycin and para-aminosalicylic acid. In treated cases, 86.8% were found to have some resistance to one or more drugs. Overall, resistance to streptomycin was found to be commonest. Drug resistance was observed to be somewhat less common in extrapulmonary than in pulmonary tuberculosis, with streptomycin resistance predominating.
102
SIDDIQI, STAUFFER, ALI AND MIDDLEBROOK
MATERIALS AND METHODS
Selection of patients. Initially a request was made to all physicians and surgeons in major hospitals in Lahore for cooperation in this study. The patients who reported to these hospitals were examined and diagnosed in the hospital and then referred to us for further examination. All the surgical cases were operated on in the hospital and the pathologic specimens were collected by our representative who also carried out further investigations. On each study case, a history was taken and tuberculin tests were performed using PPD-S and PPD-like antigens from atypical mycobacteria. During interviews of the patients, special emphasis was given to the investigation of every history of previous medications. The patients were divided into two main groups according to the reported site of disease and then subdivided into different categories according to their history of previous therapy. Group 1: Pulmonary tuberculosis, diagnosed by relevant history and chest roentgenogram. A) Untreated cases: Only those patients who gave no history of previous treatment or those who had chemotherapy for not more than 10 days were included in this group. From more than 3000 new patients interviewed, 260
were selected who met this definition of untreated cases. Care was taken not to include in this group those patients who did not seem sure of their past history or who had had treatment for more than 10 days. Of these 260 untreated cases, 130 were culture positive; a total of 100 randomly selected isolates were studied for drug susceptibility in vitro B) Treated cases: Sputum specimens from 270 patients who gave a history of taking anti-tuberculous drugs for more than 10 days were processed for culture. Most of these patients had taken more than one primary drug for long periods. Out of 121 culturepositive cases, 68 randomly selected sputum specimens were studied for drug susceptibility. Group 2: Extrapulmonary tuberculosis: A total of 946 extrapulmonary specimens from patients newly diagnosed by presenting symptoms, signs and histories were processed for culture of mycobacteria. A total of 262 cases were culture positive for mycobacteria including "atypicals." Of these, 162 randomly selected isolates of M. tuberculosis were tested for drug susceptibility. (Those specimens which yielded atypical mycobacteria were included in a separate study and will not be dealt with here). Positive cultures were divided into three categories according to the treatment history of the patients. A) Untreated cases: Patients having no history of anti-tuberculous therapy or those who had chemotherapy for not more than 10 days. B) Treated cases: Patients having a definite history of anti-tuberculous treatment for more than 10 days. C) Doubtful cases: Patients having uncertain histories of treatment. Processing of specimens. From each patient approximately 10 ml of sputum specimen was collected in a sterile, disposable, screw-capped tube. For digestion and de-
Downloaded from https://academic.oup.com/aje/article-abstract/103/1/101/151952 by Tulane University user on 18 January 2019
eral, including tests for catalase activity as well as drug susceptibilities of the strains isolated. Opportunity was provided in Pakistan, in an area where the incidence of both pulmonary and extrapulmonary tuberculosis is still high, to collect and study a large number of pathologic specimens from patients with both forms of disease, excluding mycobacterial species other than M. tuberculosis, and by standard, modern bacteriologic methods including catalase testing. This report describes the results of such studies in the Lahore area during 1968-1972.
EPIDEMIOLOGY OF TUBERCULOSIS
103
Downloaded from https://academic.oup.com/aje/article-abstract/103/1/101/151952 by Tulane University user on 18 January 2019
contamination of the specimen, sodium jected to the direct drug susceptibility hydroxide-N-acetyl-L-cysteine was used as testing with only one concentration of the described by Kubica et al. (19). (This primary drugs; i.e., INH 0.2 Mg/ml of 7H-10 process has some superiority over other agar, streptomycin 2.0 Mg/ml and PAS 2.0 methods because there is less lethal effect Mg/ml. A moderately turbid bacterial suson mycobacteria due to a lower concentra- pension was prepared comparable to the tion of sodium hydroxide). After treatment MacFarland Number One Standard and with equal volumes of the specimen and equivalent to approximately 1 mg bacterial sodium hydroxide-N-acetyl-L-cysteine so- cells/ml of suspension. Two to three sdilulution the suspension was centrifuged at tionss of this suspension, generally 10" and 3000 rpm for 20-30 minutes; the superna- 10" , were inoculated on duplicate drug tant was poured off and the sediment was quadrant plates using three drops from used for making smears and inoculating Pasteur pipette for each quadrant (approximately 0.03 ml/drop). All the drug plates culture media. In the handling of surgical specimens, a were read after 30 days. Growth of mycosmall portion was saved for histopathology bacteria on the quadrants was reported as while the remainder of the specimen was follows: macerated in a teflon grinder, suspended in Confluent (>500 colonies) 200-500 colonies about 10 ml sterile phosphate buffer and 100-200 colonies then handled according to the same stan50-100 colonies dard routine as used for the sputum speci
Vol. 103, No
Copyright © 1976 by The Johns Hopkins University School of Hypene and Public Health
1
Printed mil SA
SOME BACTERIOLOGIC ASPECTS OF THE EPIDEMIOLOGY OF PULMONARY AND EXTRAPULMONARY TUBERCULOSIS1 2 SALMAN H. SIDDIQI, JOHN C. STAUFFER, MIR ABID ALI AND GARDNER MIDDLEBROOK
Although both catalase-positive and catalase-negative isoniazid-resistant strains of M. tuberculosis were isolated from patients with pulmonary disease, no catalase-negative strains were isolated from patients with extrapulmonary disease, suggesting limited pathogenic potentialities of catalase-negative strains for man. Epidemiologic aspects of these observations are discussed. catalase; drug resistance, microbial; tuberculosis; virulence INTRODUCTION
Numerous studies have been reported on drug resistance to tubercle bacilli (MycoReceived for publication July 31, 1975. Abbreviation INH, isoniazid, PAS, para-aminosalicylic acid, PPD-S, purified protein derivative of
bacterium tuberculosis) from patients in United States (1-10), the United Kingd o m (n~1^' a n d i n P a r t s o f t h e w o r l d where the incidence of tuberculosis is still very high (14-18). These studies have been devoted almost exclusively to pulmonary
t h e
,
. .
• ,
•• ,
P
tuberculin-standard tuberculosis, with little concern for ex1 From the Pakistan Medical Research Center, trapulmonary forms of the disease, probaUniversity of Maryland International CenterforMed- b j b e c a u s e human-to-human transmisical Research & Training, 6, Birdwood Road, Lahore, s l 0 n . , , Pakistan, and the Department of Pathology, Univer»S by the aerogenic route from cases sity of Maryland School of Medicine, Baltimore, MD with respiratory tract lesions. No report 21 ^1. , , , , , T - „ ,,. has been cited in the literature which deals This study was supported by the US Public ., Health Service, PL-480 Grant 08-009-1, Mycobacte- w l t n a comparative study of pulmonary nal Diseases in Pakistan. and extrapulmonary tuberculosis in gen101
Downloaded from https://academic.oup.com/aje/article-abstract/103/1/101/151952 by Tulane University user on 18 January 2019
Siddiqi. S. H . J . C. Stauffer, M. Ali and G. Middlebrook (Dept. Pathology, U. of Maryland School of Medicine. Baltimore. M D 21201). Some bacteriologic aspects of the epidemiology of pulmonary and extrapulmonary tuberculosis. Am J Epidemiol 103: 101-111, 1976. A study was carried out to investigate the drug resistance patterns of the prevalent tubercle bacilli in pulmonary and extrapulmonary tuberculosis in and about the city of Lahore, Pakistan. This report includes 168 strains of Mycobacterium tuberculosis isolated from the same number of pulmonary tuberculosis cases (100 untreated cases, defined as patients either having no history of anti-tuberculous therapy or having had chemotherapy for not more than 10 days; 68 treated, defined as having had chemotherapy for more than 10 days), and 162 strains from the same number of extrapulmonary tuberculosis cases (77 untreated, 38 treated and 47 doubtful). The proportion method of drug susceptibility assay was employed. According to the procedures used in this study and with 1 % as the critical proportion for resistance, bacterial resistance was found to be very prevalent in pulmonary tuberculosis. Even among those cases in which no history of previous treatment was elicited, 46% were found to be excreting populations of tubercle bacilli having some degree of resistance to one or more of the primary drugs—isoniazid, streptomycin and para-aminosalicylic acid. In treated cases, 86.8% were found to have some resistance to one or more drugs. Overall, resistance to streptomycin was found to be commonest. Drug resistance was observed to be somewhat less common in extrapulmonary than in pulmonary tuberculosis, with streptomycin resistance predominating.
102
SIDDIQI, STAUFFER, ALI AND MIDDLEBROOK
MATERIALS AND METHODS
Selection of patients. Initially a request was made to all physicians and surgeons in major hospitals in Lahore for cooperation in this study. The patients who reported to these hospitals were examined and diagnosed in the hospital and then referred to us for further examination. All the surgical cases were operated on in the hospital and the pathologic specimens were collected by our representative who also carried out further investigations. On each study case, a history was taken and tuberculin tests were performed using PPD-S and PPD-like antigens from atypical mycobacteria. During interviews of the patients, special emphasis was given to the investigation of every history of previous medications. The patients were divided into two main groups according to the reported site of disease and then subdivided into different categories according to their history of previous therapy. Group 1: Pulmonary tuberculosis, diagnosed by relevant history and chest roentgenogram. A) Untreated cases: Only those patients who gave no history of previous treatment or those who had chemotherapy for not more than 10 days were included in this group. From more than 3000 new patients interviewed, 260
were selected who met this definition of untreated cases. Care was taken not to include in this group those patients who did not seem sure of their past history or who had had treatment for more than 10 days. Of these 260 untreated cases, 130 were culture positive; a total of 100 randomly selected isolates were studied for drug susceptibility in vitro B) Treated cases: Sputum specimens from 270 patients who gave a history of taking anti-tuberculous drugs for more than 10 days were processed for culture. Most of these patients had taken more than one primary drug for long periods. Out of 121 culturepositive cases, 68 randomly selected sputum specimens were studied for drug susceptibility. Group 2: Extrapulmonary tuberculosis: A total of 946 extrapulmonary specimens from patients newly diagnosed by presenting symptoms, signs and histories were processed for culture of mycobacteria. A total of 262 cases were culture positive for mycobacteria including "atypicals." Of these, 162 randomly selected isolates of M. tuberculosis were tested for drug susceptibility. (Those specimens which yielded atypical mycobacteria were included in a separate study and will not be dealt with here). Positive cultures were divided into three categories according to the treatment history of the patients. A) Untreated cases: Patients having no history of anti-tuberculous therapy or those who had chemotherapy for not more than 10 days. B) Treated cases: Patients having a definite history of anti-tuberculous treatment for more than 10 days. C) Doubtful cases: Patients having uncertain histories of treatment. Processing of specimens. From each patient approximately 10 ml of sputum specimen was collected in a sterile, disposable, screw-capped tube. For digestion and de-
Downloaded from https://academic.oup.com/aje/article-abstract/103/1/101/151952 by Tulane University user on 18 January 2019
eral, including tests for catalase activity as well as drug susceptibilities of the strains isolated. Opportunity was provided in Pakistan, in an area where the incidence of both pulmonary and extrapulmonary tuberculosis is still high, to collect and study a large number of pathologic specimens from patients with both forms of disease, excluding mycobacterial species other than M. tuberculosis, and by standard, modern bacteriologic methods including catalase testing. This report describes the results of such studies in the Lahore area during 1968-1972.
EPIDEMIOLOGY OF TUBERCULOSIS
103
Downloaded from https://academic.oup.com/aje/article-abstract/103/1/101/151952 by Tulane University user on 18 January 2019
contamination of the specimen, sodium jected to the direct drug susceptibility hydroxide-N-acetyl-L-cysteine was used as testing with only one concentration of the described by Kubica et al. (19). (This primary drugs; i.e., INH 0.2 Mg/ml of 7H-10 process has some superiority over other agar, streptomycin 2.0 Mg/ml and PAS 2.0 methods because there is less lethal effect Mg/ml. A moderately turbid bacterial suson mycobacteria due to a lower concentra- pension was prepared comparable to the tion of sodium hydroxide). After treatment MacFarland Number One Standard and with equal volumes of the specimen and equivalent to approximately 1 mg bacterial sodium hydroxide-N-acetyl-L-cysteine so- cells/ml of suspension. Two to three sdilulution the suspension was centrifuged at tionss of this suspension, generally 10" and 3000 rpm for 20-30 minutes; the superna- 10" , were inoculated on duplicate drug tant was poured off and the sediment was quadrant plates using three drops from used for making smears and inoculating Pasteur pipette for each quadrant (approximately 0.03 ml/drop). All the drug plates culture media. In the handling of surgical specimens, a were read after 30 days. Growth of mycosmall portion was saved for histopathology bacteria on the quadrants was reported as while the remainder of the specimen was follows: macerated in a teflon grinder, suspended in Confluent (>500 colonies) 200-500 colonies about 10 ml sterile phosphate buffer and 100-200 colonies then handled according to the same stan50-100 colonies dard routine as used for the sputum speci
